Molecular Engineering Of Cyclodextrin Glycosyltransferase For Glycosyl L-ascorbic Acid Synthesis

2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G), one of L-ascoribc acid (L-AA)derivatives, is prepared by transferring a glycosyl residue from α-1,4-glucan to the C2position of L-AA. AA-2G overcomes the extreme instability of L-AA in aqueous solutionespecially under particular oxidative conditions such as heat, light and metal ions, improvesthe stability in aqueous solution. Nowadays biological transformation is the main approachesfor AA-2G synthesis and cyclodextrin glycosyltransferase (CGTase) is generally consideredto be the most effective biological catalyst. Usually, α-and β-cyclodextrin are used asglycosyl donors for AA-2G production with CGTase as the catalyst because of their hightransformation efficiencies. However, neither is suitable for large-scale production of AA-2Gowing to the high cost of α-cyclodextrin and low solubility of β-cyclodextrin in aqueoussolution. To find a cheap and easily soluble substrate replacing α-and β-cyclodextrin forAA-2G industrial production is the important future development direction.In this study, molecular modification was performed on the CGTase from Paenibacillusmacerans to improve its specificity for maltodextrin and soluble starch, both of which wereregarded as cheap and easily soluble glycosyl donor for AA-2G synthesis. The main researchcontents are listed as follows:1. We obtained the cgt gene encoding α-CGTase from P. macerans JFB05-01by polymerasechain reaction (PCR) with the genome of P. macerans JFB05-01as the template. Then thecgt gene was cloned into the pET-20b(+) victor, and the expression plasmidpET-20b(+)/cgt was constructed and transformed into the host E. coli BL21(DE3). Afterexpression and purification, we got the purified CGTase which was approximately75kDa.2. Site-saturation engineering of Lys47in CGTase was performed with pET-20b(+)/cgt asthe template. When using maltodextrin as glycosyl donor, four mutants K47F, K47L,K47V and K47W showed higher AA-2G yield as compared with that produced by thewild-type CGTase. The enzymatic characteristics suggested that their optimal temperature,pH and substrate ratio were36oC, pH5.5and maltodextrin/L-AA=1/1, respectively. Attheir optimal conditions, K47V，K47F，K47L and K47W got their highest AA-2G yieldsat8,12,24and12h, respectively. The highest AA-2G titer produced by K47L was1.97g L-1, which increased64%than the wild type CGTase (WT). The reaction kineticsanalysis showed that the Km(maltodextrin) of the four mutants were lower than the WTCGTase while the Kcat/Kmof them were higher than the WT. It was also found thatcompared with the WT, the four mutants had relatively lower cyclization activities andhigher disproportionation activities. Finally, the possible reasons were further explored bystructure modeling of the mutant CGTases.3. Combination site-saturation engineering was performed on Tyr260, Tyr195and Asn265of the CGTase and seven positive mutants Y195S，Y260R，Q265K，Y260R/Q265K，Y260R/Y195S， Q265K/Y195S and Y260R/Q265K/Y195S were obtained. After purification, the highest AA-2G titer produced by Y260R/Q265K/Y195S withmaltodextrin as the glycosyl donor was1.88g L-1, which increased60%than WT. Theenzymatic characteristics suggested that their optimal temperature were36oC. And for theoptimal pH: Y260R/Q265K and Y260R/Q265K/Y195S were pH6.5, Y260R and Q265Kwere pH5.0, Y195S and Y260R/Y195S were pH5.5, Q265K/Y195S was pH7.0. Thereaction kinetics analysis showed that the affinity and catalytic efficiency of the sevenmutants towards maltodextrin were higher than the WT. It was also found that comparedwith the WT, the four mutants had relatively lower cyclization activities and higherdisproportionation activities. Finally, the possible reasons were further explored bystructure modeling of the mutant CGTases.4. Iterative saturation mutagenesis (ISM) was performed on-6subsite of the CGTase toimprove the maltodextrin specificity for AA-2G synthesis. Four positive mutants Y167S,Y167S/G179K， Y167S/G179K/G193R and Y167S/G179K/G193R/G180R wereobtained and purified. The enzymatic characteristics showed that the optimal temperatureof the mutants was28oC (different from WT), and the optimal pH were also differentfrom WT. The highest AA-2G titer produced by Y167S/G179K/G193R/G180R withmaltodextrin as the glycosyl donor was2.12g L-1, which increased84%than WT. Thereaction kinetics analysis showed that the affinity ability towards maltodextrin of the fourmutants was higher than WT. It was also found that compared with the WT, the fourmutants had relatively lower cyclization activities and higher disproportionation activities.Finally, the possible reasons were further explored by structure modeling of the mutantCGTases.5. Two chimeric enzymes CGT-CBMAmyand CGT△E-CBMAmywere obtained by fusing acarbohydrate-binding module (CBM) from Alkalimonas amylolytica α-amylase (CBMAmy)to cyclodextrin glycosyltransferase (CGTase) from P. macerans. The enzymaticcharacteristics showed that when using soluble starch as the glycosyl donor for AA-2Gsynthesis, the optimal temperature and pH of the two chimeric enzymes were28oC andpH6.5. The highest AA-2G titers of CGT-CBMAmyand CGT△E-CBMAmywere3.94-and5.94-fold of yield from WT. The reaction kinetics analysis showed that the affinity abilityof the two chimeric enzymes towards soluble starch improved. Compared to WT, the twofusion enzymes had relatively high hydrolysis and disproportionation activities. Finally,the possible reasons were further explored by structure modeling of the mutant CGTases.6. Fuse six self-assembling amphipathic peptides (SAPs) to the N-terminal of CGTase toimprove AA-2G synthesis with soluble starch as the glycosyl donor. The enzymaticcharacteristics showed that all fusion enzymes have lower cyclization activities than WT.Compared to WT, SAP5-CGTase and SAP6-CGTase increased the disproportionationactivities, and the AA-2G titers produced by them increased about1.33-and2.36-fold.The reaction kinetics analysis showed that the affinity ability of the two fusion enzymestowards soluble starch improved compared to WT. Finally, the possible reasons werefurther explored by structure modeling of the mutant CGTases.